This invention relates to thermally stabilized freeze-dried vesicle containing ultrasound contrast agents and a process for their preparation.
Vesicles (the term is used herein to denote unilamellar and multilamellar structures, eg. structures referred to variously as liposomes, micelles, microbubbles and micro-balloons) are frequently used as a means for delivering therapeutically or diagnostically active agents. In the field of ultrasound imaging contrast media, vesicles containing materials (herein referred to as vesicular materials) which are gaseous at body temperatures may be used as echogenic contrast agents, particularly for administration into the vasculature.
Vesicular contrast media will generally be administered in the form of an aqueous dispersion containing a low concentration of the vesicles relative to the aqueous carrier medium. Accordingly storage and transportation of such vesicular contrast agents is made significantly more efficient if the vesicles can be stored in a dried form.
Freeze-drying of vesicular compositions is possible and, for this, formulation excipients are generally included in the composition to aid the drying technique. Such excipients generally serve one of two functions. Bulking agents are added to increase the total solids content in order to achieve a mechanically more robust product. Stabilizers, otherwise referred to as cryoprotectants or lyoprotectants, are added to aid the formation of the glassy state produced during dehydration and to provide physical strength in the dried product. Examples of stabilizers used in this way include mannitol and glucose.
Freeze-dried vesicular ultrasound contrast agents while providing advantages for transport and storage due to the reduction in bulk relative to the aqueous ready-to-use dispersions, also provide problems since the freeze-dried product is not thermally stable in the range of ambient temperatures normally encountered during transportation and storage and as a result must be maintained, prior to secondary production, in an environment in which the temperature is maintained below ambient (eg. at 5 to 10xc2x0 C.).
It has now surprisingly been found that by appropriate choice of the stabilizer used for freeze-drying it is possible to produce freeze-dried vesicular ultrasound contrast agents which are thermally stable at ambient temperatures and above, and indeed at all temperatures normally encountered during transportation and storage.
The thermally stable freeze-dried product may then be stored and transported without need of temperature control of its environment and in particular may be supplied to hospitals and physicians for on site formulation into an administrable dispersion without requiring such users to have special storage facilities.
Thus viewed from one aspect the invention provides a freeze-dried vesicle containing ultrasound contrast agent containing a freeze-drying stabilizer and thermally stable at temperatures in excess of 20xc2x0 C., preferably at least 22xc2x0 C., especially 22xc2x0 C., at least 25xc2x0 C., more preferably at least 30xc2x0 C. and especially preferably at least 40xc2x0 C., eg. up to 65xc2x0 C. or higher. Alternatively viewed the invention provides a freeze-dried vesicle containing ultrasound contrast agent containing a freeze-drying stabilizer and having a glass transition temperature (Tg) above 20xc2x0 C., preferably 22xc2x0 C., especially preferably at least 25xc2x0 C., more preferably at least 30xc2x0 C. and especially preferably at least 40xc2x0 C., eg. up to 65xc2x0 C. or higher.
Viewed from a further aspect the invention provides a process for the preparation of a thermally stable freeze-dried vesicle containing ultrasound contrast agent, which process comprises freeze-drying an aqueous dispersion comprising a vesicular ultrasound contrast agent and a freeze-drying stabilizer or mixture of stabilizers, characterised in that said stabilizer or mixture of stabilizers has a Tg value of at least 20xc2x0 C. (preferably at least 22xc2x0 C., especially at least 25xc2x0 C., more preferably at least 30xc2x0 C. and especially preferably at least 40xc2x0 C., eg. up to 65xc2x0 C. or higher) and a Tgxe2x80x2 value of xe2x88x9237xc2x0 C. or above (preferably above xe2x88x9236xc2x0 C., especially preferably above xe2x88x9235xc2x0 C., eg. xe2x88x9210 to xe2x88x9237xc2x0 C.).
Viewed from a still further aspect the invention provides an ultrasound contrast medium comprising an aqueous carrier medium, a freeze-drying stabilizer or mixture of stabilizers and an echogenic vesicular ultrasound contrast agent, characterised in that said stabilizer or mixture of stabilizers has a Tg value of at least 20xc2x0 C., (preferably at least 22xc2x0 C., especially at least 25xc2x0 C., more preferably at least 30xc2x0 C. and especially preferably at least 40xc2x0 C., eg. up to 65xc2x0 C. or higher) and a Tgxe2x80x2 value of xe2x88x9237xc2x0 C. or above (preferably above xe2x88x9236xc2x0 C., especially preferably above xe2x88x9235xc2x0 C., eg. xe2x88x9210 to xe2x88x9237xc2x0 C.).
Viewed from a yet still further aspect the invention provides the use of a freeze-drying stabilizer or mixture of stabilizers having a Tg value of at least 20xc2x0 C., (preferably at least 22xc2x0 C., especially at least 25xc2x0 C., more preferably at least 30xc2x0 C. and especially preferably at least 40xc2x0 C., eg. up to 65xc2x0 C. or higher) and a Tgxe2x80x2 value of xe2x88x92370xc2x0 C. or above (preferably above xe2x88x9236xc2x0 C., especially preferably above xe2x88x9235xc2x0 C., eg. xe2x88x9210 to xe2x88x9237xc2x0 C.) for the manufacture of a vesicle containing ultrasound contrast medium for use in diagnosis involving diagnostic ultrasound imaging.
Viewed from a further aspect the invention provides a process for the storage or transportation of a vesicular ultrasound contrast agent, characterized in that said agent is in freeze-dried form, contains a freeze-drying stabilizer and has a glass transition temperature (Tg) of at least 20xc2x0 C. (preferably at leat 22xc2x0 C., etc.), and in that storage and transportation takes place without cooling.
Viewed from a still further aspect the invention provides a process for the preparation of a vesicle containing ultrasound contrast medium, said process comprising dispersing a freeze-dried contrast agent according to the invention in physiologically tolerable aqueous dispersion medium.
For any material, Tg is the glass transition temperature of the dried material while Tgxe2x80x2 is the glass transition temperature of the maximally freeze-concentrated pure aqueous solution of the material.
Besides the improved thermal stability, the freeze-dried vesicular contrast agents according to the invention also surprisingly enhance the ability of the vesicles to retain the halocarbon gases and gas precursors commonly used in ultrasound contrast agents.
In the invention, the ultrasound contrast agent may be any physiologically tolerable echogenic vesicular agent, preferably however the vesicles will contain a gas or gas precursor (eg. a compound or compound mixture which is substantially in gaseous (including vapour) form at normal human body temperatures (37xc2x0 C.)). Any biocompatible gas, gas precursor or mixture may be employed. The gas may thus, for example, comprise air; nitrogen; oxygen; carbon dioxide; hydrogen; nitrous oxide; an inert gas such as helium, argon, xenon or krypton; a sulphur fluoride such as sulphur hexafluoride, disulphur decafluoride or trifluoromethylsulphur pentafluoride; selenium hexafluoride; an optionally halogenated silane such as tetramethylsilane; a low molecular weight hydrocarbon (e.g. containing up to 7 carbon atoms), for example an alkane such as methane, ethane, a propane, a butane or a pentane, a cycloalkane such as cyclobutane or cyclopentane, an alkene such as propene or a butene, or an alkyne such as acetylene; an ether; a ketone; an ester; a halogenated low molecular weight hydrocarbon (e.g. containing up to 7 carbon atoms); or a mixture of any of the foregoing. At least some of the halogen atoms in halogenated gases advantageously are fluorine atoms. Thus biocompatible halogenated hydrocarbon gases may, for example, be selected from bromochlorodifluoro-methane, chlorodifluoromethane, dichlorodifluoromethane, bromotrifluoromethane, chlorotrifluoromethane, chloropentafluoroethane, dichlorotetrafluoroethane and perfluorocarbons, e.g. perfluoroalkanes such as perfluoromethane, perfluoroethane, perfluoropropanes, perfluorobutanes (e.g. perfluoro-n-butane, optionally in admixture with other isomers such as perfluoro-iso-butane), perfluoropentanes, perfluorohexanes and perfluoroheptanes; perfluoroalkenes such as perfluoropropene, perfluorobutenes (e.g. perfluorobut-2-ene) and perfluorobutadiene; perfluoroalkynes such as perfluorobut-2-yne; and perfluorocycloalkanes such as perfluorocyclobutane, perfluoromethylcyclobutane, perfluorodimethylcyclobutanes, perfluorotrimethyl-cyclobutanes, perfluorocyclopentane, perfluoromethyl-cyclopentane, perfluorodimethylcyclopentanes, perfluorocyclohexane, perfluoromethylcyclohexane and perfluorocycloheptane. Other halogenated gases include fluorinated, e.g. perfluorinated, ketones such as perfluoroacetone and fluorinated, e.g. perfluorinated, ethers such as perfluorodiethyl ether.
Particularly preferably, the vesicles will contain a perfluoroalkane, especially a perfluorobutane, perfluoropentane or perfluorohexane, in particular n-perfluorobutane.
In the vesicles, the membrane may be formed of any physiologically tolerable membrane forming material, in particular phospholipids, and may be cross-linked or non-cross-linked. Membranes formed of mixtures of charged and non-charged phospholipids are especially preferred and it is particularly preferred that the vesicles should carry a net surface charge, preferably a negative charge. Such phospholipid vesicles have particularly favourable blood residence times.
The vesicles may further be provided with a blood residence prolonging agent, eg. by conjugating such an agent to the membrane or to a lipophilic group which will anchor within the membrane. Such blood residence prolonging agents, eg. polyalkylene oxides such as polyethylene glycol, can act as opsonisation inhibitors delaying the uptake of the vesicles from the vasculature by the reticuloendothelial system.
Desirably at least 75%, preferably substantially all of the phospholipid material in the contrast agents of the invention consists of molecules which individually bear a net overall charge under conditions of preparation and/or use, which charge may be positive or, more preferably, negative. Representative positively charged phospholipids include esters of phosphatidic acids such as dipalmitoylphosphatidic acid or distearoylphosphatidic acid with aminoalcohols such as hydroxyethylenediamine. Examples of negatively charged phospholipids include naturally occurring (e.g. soya bean or egg yolk derived), semisynthetic (e.g. partially or fully hydrogenated) and synthetic phosphatidylserines, phosphatidylglycerols, phosphatidylinositols, phosphatidic acids and cardiolipins. The fatty acyl groups of such phospholipids will typically each contain about 14-22 carbon atoms, for example as in palmitoyl and stearoyl groups. Lyso forms of such charged phospholipids are also useful in accordance with the invention, the term xe2x80x9clysoxe2x80x9d denoting phospholipids containing only one fatty acyl group, this preferably being ester-linked to the 1-position carbon atom of the glyceryl moiety. Such lyso forms of charged phospholipids may advantageously be used in admixture with charged phospholipids containing two fatty acyl groups.
Phosphatidylserines represent particularly preferred phospholipids for use in contrast agents according to the invention and preferably constitute a substantial part, e.g. at least 80% of the initial phospholipid content thereof, for example 85-92%, although this may subsequently be reduced somewhat, e.g. to ca. 70%, in subsequent processing such as heat sterilisation. While we do not wish to be bound by theoretical considerations, it may be that ionic bridging between the carboxyl and amino groups of adjacent serine moieties contributes to the stability of such systems. Preferred phosphatidylserines include saturated (e.g. hydrogenated or synthetic) natural phosphatidylserine and synthetic or semisynthetic dialkanoylphosphatidylserines such as distearoyl-phosphatidylserine, dipalmitoylphosphatidylserine and diarachidoylphosphatidylserine.
An important advantage of the use of such phosphatidylserine-based contrast agents is that the body recognises aged red blood cells and platelets by high concentrations of phosphatidylserine on their surface and so may eliminate such contrast agents from the blood stream in a manner similar to the elimination of aged red blood cells. Furthermore, since the surface of such contrast agents may be registered as endogenous by the body, they may avoid induction of adverse systemic side effects such as haemodynamic effects and other anaphylactic reactions which may accompany administration of some liposome preparations (see e.g. WO-A-95/12386).
Liposomal ultrasound contrast agents suitable for use according to the invention may be prepared as described in the literature, see for example WO-A-92/22247, WO-A-94/28780, WO-A-93/05819, WO-A-95/16467, PCT/GB96/01361 and Unger et al. Invest. Radiol. 29 (Suppl. 2): S134-S136 (1994).
The stabilizer used according to the invention may be a physiologically tolerable freeze-drying stabilizer (or mixture) having a glass transition temperature (Tg) above 20xc2x0 C., eg. in the range 25 to 70xc2x0 C., and having a Tgxe2x80x2 value of xe2x88x9237xc2x0 C. or above. Examples of suitable stabilizers include sucrose, maltose H2O, trehalose, raffinose and stachyose. One particularly suitable example is sucrose, optionally in admixture with minor quantities (eg. up to 20% by weight, preferably up to 10% by weight) of other stabilizers.
In general the stabilizer will be present in the composition being freeze-dried at a concentration significantly in excess of that of the vesicular contrast agent, eg. at a weight ratio of at least 10:1, more normally at least 20:1, optimally as high as 200:1, eg. up to 5000:1 or even higher. Accordingly, the contribution to the glass transition temperature (Tg) of the dried product is relatively independent of the vesicular component and candidate stabilizers can be screened easily by routine techniques to determine whether they, in combination with the other excipients present in the aqueous carrier medium, dry to form a product having a glass transition temperature (Tg) above 20xc2x0 C.
Conveniently the stabilizer will be present at 1 to 50% by weight, preferably 5 to 30%, more especially about 10 to 20% by weight, in the composition undergoing freeze-drying. The concentration of stabilizer may if desired be well in excess of isotonic concentrations since, on reconstitution after freeze-drying, the product can be diluted. The vesicle component will preferably be present at 0.01 to 5% by weight, preferably 0.1 to 3%, especially preferably about 0.5 to 1.5% by weight (considering its weight to be only the weight of the membrane forming material). The quantity of stabilizer relative to the reconstitution fluid used to transform the freeze-dried product into an administrable dispersion will be selected dependent on the body region or organ to be imaged and on the administration mode chosen. By way of example it may be at least twice that in the composition which underwent freeze-drying.
For ultrasound applications such as echocardiography, in order to permit free passage through the pulmonary system and to achieve resonance with the preferred imaging frequencies of about 0.1-15 MHz, it may be convenient to employ vesicles having an average size of 0.1-10 xcexcm, e.g. 1-7 xcexcm. The vesicles may be produced with a very narrow size distribution within the range preferred for echocardiography, thereby greatly enhancing their echogenicity as well as their safety in vivo, and rendering the contrast agents of particular advantage in applications such as blood pressure measurements, blood flow tracing and ultrasound tomography. Thus, for example, products in which over 90% (e.g. at least 95%, preferably at least 98%) of the vesicles have diameters in the range 1-7 xcexcm and less than 5% (e.g. not more than 3%, preferably not more than 2%) of the vesicles have diameters above 7 xcexcm may readily be prepared.
In ultrasound applications the contrast media may, for example, be administered in doses such that the amount of membrane forming material (eg. phospholipid) injected is in the range 0.1-10 xcexcg/kg body weight, more preferably 1-5 xcexcg/kg. It will be appreciated that the use of such low levels of membrane forming material is of substantial advantage in minimising possible toxic side effects.
The overall membrane forming material concentration in ready-to-use compositions made using the dried product of the invention will desirably be in the range 0.01 to 5% by weight, preferably 0.05 to 2.0% and particularly about 0.5% by weight.
The composition subjected to freeze-drying will advantageously contain at least one bulking agent, eg. a polyol (eg. a C3 polyol such as glycerol or propylene glycol) or a polysaccharide such as dextran, or a polyglycol such as polyethylene glycol or mixtures thereof. Typically the bulking agent may be used in concentrations similar to or slightly less than that of the stabilizer, eg. 3 to 10% by weight, preferably about 5% by weight. The bulking agents should be able to crystallize during the freeze-drying process as only in this state do they have a neutral effect on product stability. They are thus distinguished from the stabilizers which should be present in the amorphous state during freeze-drying.
Other excipients may if desired by present in the composition being dried or may be added on formulation for administration. Such excipients may for example include pH regulators, osmolality adjusters, viscosity enhancers, emulsifiers, etc. and may be used in conventional amounts.
The dried product will generally be in powder form and is readily reconstitutable in water, an aqueous solution such as saline (which may advantageously be balanced so that the final product for injection is not hypotonic), or a solution of one or more tonicity adjusting substances such as salts (eg. of plasma cations with physiologically tolerable counterions), or sugars, sugar alcohols, glycols and other non-ionic polyol materials (eg. glucose, sucrose, sorbitol, mannitol, glycerol, polyethylene glycols, propylene glycols and the like). Reconstitution will generally require only minimal agitation such as may, for example, be provided by gentle hand-shaking. The size of the vesicles so generated is consistently reproducible and in practice is independent of the amount of agitational energy applied, being determined by the size of the vesicles formed in the initial vesicle dispersion, this size parameter surprisingly being substantially maintained in the lyophilised and reconstituted product. Thus, since the size of the vesicles in the initial dispersion may readily be controlled by process parameters such as the method, speed and duration of agitation, the final vesicle size may readily be controlled.
The volume and concentrations of the reconstitution liquid may desirably be balanced to make the resulting ready-to-use formulations substantially isotonic. Hence the volume and concentration of reconstitution fluid chosen will be dependent on the type and amount of stabilizer (and other bulking agents) present in the freeze-dried product.
Lyophilised products according to the invention have proved to be storage stable for several months under ambient conditions. The vesicle dispersions generated upon reconstitution in water (or other reconstitution liquids as discussed above) may be stable for considerable lengths of time, eg. up to at least 12 hours, permitting considerable flexibility as to when the dried product is reconstituted prior to injection.
If the reconstitution liquid contains as a tonicity adjuster the same compound as is used as a stabilizer in the lyophilization, the amount of stabilizer present in the composition for freeze-drying need only be sufficient to give the optimal stabilization during freeze-drying. Isotonicity of the final product may thus be obtained by selecting an adequate amount and concentration of the reconstitution liquid. Hence a considerable flexibility is permitted as to the concentration and type of compound(s) to be used as stabilizer(s) during the freeze-drying step, and concentration and type of compound(s) in the reconstitution liquid, while still achieving a stable reconstituted product.
Freeze-drying according to the invention may be effected in a conventional manner although using stabilizers according to the invention may have the added advantage that, since the compositions before drying generally have higher glass temperatures (Tgxe2x80x2) than equivalent compositions containing cryoprotectants such as glucose or mannitol, shorter freeze-drying cycles may be used.
The invention has been described above with reference to vesicular ultrasound contrast agents. However it is also applicable to vesicular contrast agents for other diagnostic imaging modalities (eg. MRI, X-ray, SPECT, PET, magnetographic imaging etc.).